Process for the purification of serum albumin

ABSTRACT

Recombinantly produced serum albumin is purified in a series of steps, optionally by incubation with an anion-exchange adsorbent, followed by affinity chromatography employing a hydrophobic solid phase and using a water-soluble lipid anion as desorbens in the aqueous phase. Said immobile phase comprises a carrier coupled to a 2-mercapto or 2-hydroxy alkanoic acid.

INTRODUCTION TECHNICAL FIELD

[0001] The field concerns purification of serum albumins, particularlyhuman serum albumins.

BACKGROUND AND RELEVANT LITERATURE

[0002] Human serum albumin (HSA) is the major protein component ofplasma. The primary function of albumin in plasma is maintenance of thecolloid osmotic pressure within the blood vessel. Furthermore, theprotein acts as a carrier of several ligands, for instance bilirubin andfatty acids. (See reviews by F. Rothstein, V. M. Rosenoer and W. L.Hughes in Albumin Struct. Funct. Uses (1977) 7-25; U. Kragh-Hansen,Pharmacol. Rev. (1981) 33:17-53; T. Peters Jr., Adv. Prot. Chem. (1985)37:161-245).

[0003] Purified serum albumin is indicated for the prevention andtreatment of hypovolemic shock, in conditions where there is severehypoalbuminemia, as an adjunct in haemodialysis and in cardiopulmonarybypass procedures and in conjunction with exchange transfusion in thetreatment of neonatal hyperbilirubinemia.

[0004] Since large amounts of serum albumin are necessary for therapyand the source of serum albumin (plasma) is limited, other techniqueshave been sought to produce HSA in large quantities. Successes have beenreported in the production of HSA by fermentation using transformedmicroorganisms or cell lines made by recombinant DNA techniques. See,for example, EP-A-0073646.

[0005] However, one of the major problems in the purification of serumalbumin produced by fermentation using transformed cells is the presenceof contaminating components from the growth medium (fermentation broth)or cell lysate, which have to be removed in order to obtain purified,homogeneous serum albumin.

[0006] In EP-A-0361991 the purification of HSA produced with transformedyeast, using techniques known in the art, yields a product of more than99% purity. For a pharmaceutical preparation a higher purity isdesirable.

[0007] Recently a process for the purification of serum albumins basedon a process in three steps was disclosed in EP-A-0319067. This processstarting with an alkaline precipitation step followed by anion-exchangechromatography and finally affinity chromatography yields a product witha good yield and purity. In EP-A-0319067 a BrCN-activated Sepharose 4Bsupport was mentioned which was prepared according to the methoddescribed by Wichman and Andersson (1974). For industrial use theaffinity matrix based on BrCN-activated sepharose has two majordrawbacks.

[0008] First, the isourea linkage (M. Wilchek, T. Miron and J. Kohn in:Methods in Enzymology (1984) 104:3, W. B. Jakoley Ed., Academic Press,London) resulting from the reaction with the primary alkylamine (spacer)has a positive charge under physiological conditions. This chargedspacer shows anion-exchange like characteristics that might interferewith the biospecific adsorption. A second disadvantage is the limitedstability of the isourea linkage under slightly alkaline conditions (C.M. Yang and G. T. Tsao in: Ad. Biochem. Engin. (1982) 25:19, A. FiechterEd., Springer Verlag, Berlin-Heidelberg) which enables the use of thismatrix under sanitizing conditions (0.1-2.0 M NaOH) necessary for theproduction of pharmaceutical products.

[0009] Therefore, there is a great need for a practical process forlarge-scale purification of human serum albumin with a high recovery anda very high purity.

SUMMARY OF THE INVENTION

[0010] Human serum albumin, produced by a transformed host, is purifiedby ion-exchange chromatography, followed by affinity chromatographyemploying a lipophilic surface immobile phase comprising a carriercoupled to a 2-mercapto or 2-hydroxy C4-C14 alkanoic acid, or salt orester thereof. The serum albumin degradation products present at the endof the fermentation which strongly resemble the mature, intact albuminare selectively removed by the fatty acid affinity chromatographyapplied. High recovery and extremely high purity are achieved.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1: SDS-PAGE under reducing conditions (CBB R-250 stain).Molecular weight markers: lanes 1 and 5 (24 μg). Purified recombinantHSA after Q-sepharose and affinity chromatography: lane 3 (30 μg).Commercially available from human blood derived HSA (Sigma human albuminNo. A-8763: lot: 109F-9304): lane 4 (30 μg).

[0012]FIG. 2: High performance size-exclusion chromatography on BiosilTSK-250® of purified recombinant HSA.

[0013]FIG. 3: High performance size-exclusion chromatography on BiosilTSK-250® of commercially available from human blood derived HSA.

[0014]FIG. 4: High performance ion-exchange chromatography on Mono Q® ofpurified recombinant HSA after Q-sepharose and affinity chromatography.

[0015]FIG. 5: High performance ion-exchange chromatography on Mono Q® ofcommercially available from human blood derived HSA after Q-sepharoseand affinity chromatography.

[0016]FIG. 6: Isoelectrofocussing (CBB R-250 stain). Markers (lanes 1and 4), purified recombinant HSA after Q-sepharose and affinitychromatography (lane 2 (20 μg)) and commercially available from humanblood derived HSA (lane 3 (20 μg)).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0017] In accordance with the subject invention, human serum albumin,prepared by fermentation using transformed hosts, e.g. microorganisms orcell lines, made by recombinant DNA techniques, is purified with highrecovery and in high purity for use as a pharmacological product. Thepurification method is preceded by centrifugation and ultrafiltration ofa fermentation broth. The method can be applied to the thus obtainedsupernatant or to the cells after lysis. It generally involves thefollowing steps:

[0018] binding of the serum albumin under slightly acidic conditions toan anion-exchange resin; the albumin is desorbed from the resin bylowering the pH of the eluens;

[0019] next, optionally, ultrafiltration of the medium;

[0020] finally, the essential chromatographic step consisting ofaffinity chromatography employing a lipophilic immobile phase composedof a hydrophilic matrix, spacer and fatty acid derivative as ligand anda lipophilic anion as desorbens in the eluens.

[0021] The serum albumin may then be harvested by desalting andconcentration. The serum albumin produced by the subject process issubstantially homogeneous and monomeric, viz. more than 99.95% pure.

[0022] The method finds use with the production of serum albumin,particularly human serum albumin, prepared by recombinant techniquesemploying microorganisms or cell lines. The microorganisms may beprokaryotic or eukaryotic, particularly eukaryotic, and include bacteriasuch as E. coli, B. subtilis, B. licheniformis, Streptomyces,Pseudomonas, etc.

[0023] Among eukaryotes are yeasts, such as Saccharomyces,Schizosaccharomyces, Kluvveromyces, Candida, etc., filamentous fungi,Neurospora, Aspergillus, etc.

[0024] The expression of the serum albumin may result in secretion orretention of the product in the organism. The broth may be removedbatch-wise or continuously from the fermenter. In the case of secretion,the cell-free supernatant is used in the purification process. Thecell-free supernatant is obtained by clarification of the fermentationbroth, conveniently by centrifuging and/or filtering the broth, usingultrafiltration for concentration of the protein product. The filterwill generally have a cut-off of 500-100,000 D, more usually at leastabout 10,000 D.

[0025] Where the product or part thereof is retained in the cytoplasm ofthe cell, the cells are harvested. A lysate may be produced inaccordance with any convenient technique, using mechanical or chemicaldisruption of the cells to produce the lysate. The cellular debris maybe removed by centrifugation.

[0026] The pH of the cell-free supernatant or cell lysate is adjusted toabout 5 to 9, more preferably to about 6 to 6.5. The pH may be modifiedby any convenient means, such as the addition of sodium hydroxide, orother convenient base, usually at a normality in the range of about 0.1to concentrated.

[0027] The next step is anion-exchange chromatography. The cell-freesupernatant with a buffer concentration from about 20 to 200 mM will beapplied to the preconditioned column. The loading of the anion gel willbe from 5 to 50 g of protein per liter gel, more preferably from 15 to25 g of protein per liter gel.

[0028] Next the gel can be washed with equilibration buffer. The volumeof the washes is not critical, generally being from 0.5-10 based on thevolume of the gel. The buffer will generally have a conductivity of0.1-100 mS/cm, preferably between about 1 to 10 mS/cm. The albumin isdesorbed from the gel by eluting with a generally dilute buffer ofmoderately low pH. The buffer concentration will be from 10 to 100 mM.Desirably the buffer will be at a pH in the range of about pH 4.0-6.0,more desirable between pH 4.25 and 4.75.

[0029] The ion-exchange step removes nucleic acids, endotoxins and themajor part of the contaminating non-albumin like proteins.Anion-exchange Adsorbents such as QAE or DEAE bound to a commerciallyavailable carrier are employed.

[0030] The next essential step is affinity chromatography, where alipophilic immobile phase is used. The binding of the lipophilicmolecules to the gel is performed by using a bifunctional reagent,capable of both activating and coupling of the lipophilic molecules.Examples of such reagent are epoxy compounds, as for instanceepihalohydrin, such as epichlorohydrin, or bisoxirans such as analkanediolether, for instance 1,4-butanedioldiglycidoxyether. Thereaction with the gel matrix yields derivatives which possess ahydrophilic, reactive epoxide which can react with nucleophilic ligandssuch as 2-mercapto or 2-hydroxy C4-C14 alkanoic acids. See for moredetails: Affinity Chromatography: A Practical Approach, ed. Dean, P. D.G., Johnson, W. S. and Middle, F. A., IRL Press, Oxford, England,ISBN-0-904147-71-1.

[0031] These affinity chromatographic adsorbents are highly stable anddo not possess a charge in the spacer.

[0032] After the ion-exchange step the pH of the medium must beincreased to physiologic conditions, normally in the range of about 6.5to 8.0, more usually in the range of about 7 to 7.5. The bufferconcentration will generally be in the range of 50 to 250 mM. Afterapplying the medium to the column the gel is washed with equilibrationbuffer. The volume of the washes is not critical, generally being from0.25-5 volume based on the volume of the gel. The buffer will generallyhave a conductivity of 1.0-20 mS/cm, preferably between about 10 to 15mS/cm. The loading of the affinity gel will be from 5-70 g of proteinper liter gel, more preferably from 15-30 g of protein per liter gel.The albumin is eluted by applying a buffer with a fatty acid with aconcentration of 25-250 mM, for instance sodium caprylate.

[0033] Besides mature human serum albumin, specific degradation productsare present in the supernatant of the fermentation broth. Thesedegradation products have a molecular weight of 40-50 kD and consist ofdomain I and II of the mature protein that comprises three domains.Domain III absent in said 40-50 kD fragment contains the principle fattyacid binding site of albumin (T. Peters, supra). Surprisingly, said40-50 kD fragments show a higher affinity for lipophilic affinity matrixthan mature human serum albumin which enables the separation of the40-50 kD fragments from the mature protein.

[0034] A wide variety of supports and adsorbents may be used as thesolid carriers or supports. Such solid carriers include inorganiccarriers, such as glass and silica gel, organic, synthetic or naturallyoccurring carriers, such as, for instance, agarose, cellulose, dextran,polyamide, polyacrylamides, vinyl copolymers of bifunctional acrylates,and various hydroxylated monomers, and the like. Commercially availablecarriers are sold under the names of Sephadex®, Sepharose®, Trisacryl®,Ultrogel®, Dynospheres®, Macrosorb®, XAD resins, and others.

[0035] The conditions for the various steps will be carried out atnon-denaturing conditions, generally at convenient temperatures in therange of about −10° C. to +30° C., more usually at about ambienttemperatures. The chromatographic steps may be performed batch-wise orcontinuously, as convenient. Any convenient method of separation may beemployed, such as centrifugation, filtration, decanting, or the like. Inthis way a preparation of isolated serum albumin with a surprisinglyhigh purity of more than 99.9% pure, particularly more than 99.95% pure,can be obtained.

[0036] All documents cited are incorporated herein by reference.

[0037] The following non-limitative examples will further illustrate theinvention.

EXAMPLES Example 1 Synthesis of Affinity Matrices

[0038] E-C10-C10:

[0039] E stands for epoxy-activated sepharose 6FF;

[0040] first C10 stands for the spacer length of 10 C atoms;

[0041] second C10 stands for the number of C atoms in the carboxylicacid ligand.

[0042] For the synthesis of the affinity matrices well known methodswere used (see e.g. Dean et al., supra).

[0043] For the preparation of C3 and C10 spacers epichlorohydrin and 1,4butanediolglycidoxyether were used, respectively.

[0044] Synthesis of the E-C10-C10 Matrix:

[0045] 1 l Sepharose 6FF was washed thoroughly with distilled water. Theexcess water was removed by a sintered glass filter. The gel wassuspended in 0.6 l of 1,4 butanediolglycidoxyether and stirred forapproximately 30 min. Next 0.6 l of a solution of 0.5 N NaOH was addedand stirring was continued for 24 h. The epoxy-activated gel wasthoroughly washed with distilled water and stored at 0° C. Yield 0.7 kgof wet product (E-C10).

[0046] The epoxy-activated sepharose 6FF (0.5 kg of wet product, E-C10)was suspended in a solution of 8 g 2-mercaptodecanoic acid in a solutionof 0.5 M sodium carbonate and stirred for 24 h. The gel was filtered ona sintered glass filter and washed successively with 0.2 M sodiumcarbonate and water. The wet gel was stored at 0° C. This matrix wascalled E-C10-C10.

[0047] The number of free carboxylic acids was determined by simpletitration and was found to be 19.3 μequivalents per gram of wet gelmatrix.

Example 2 Purification of Recombinant HSA from a Clarified FermentationBroth by Q-Sepharose and E-C10-C10-Affinity Chromatography

[0048] Fermentation:

[0049] The Kluvveromyces lactis strain CBS 2360 (Centraal Bureau voorSchimmelcultures, Baarn, The Netherlands), transformed with a plasmidcontaining the gene for HSA, was grown for 90 h at 30° C. in a mediumcontaining yeast extract 0.5% (w/v), glucose 1.4% (w/v), caseinhydrolysate 1.0% (w/v), vitamins and mineral salts. During thefermentation glucose was fed.

[0050] Filtration and Ultrafiltration:

[0051] The fermentation broth was centrifuged (5 min at 4000 rpm). Thesupernatant was filtered through an EKS filter and was concentrated 11times by ultrafiltration using a filter with a cut-off of 30,000 D. Theprotein concentration after ultrafiltration was 28 mg/ml, of which 65%is monomeric and mature recombinant HSA. The pH was adjusted to 6.4 byaddition of 0.5 M sodium acetate pH 5.5.

[0052] Q-Sepharose Chromatography:

[0053] The HSA solution was bound to a Q-sepharose FF column (load: 25 gof protein per liter sepharose), which has been equilibrated with 50 mMsodium acetate pH 5.5. After washing the gel with the equilibrationbuffer, the RHA was eluted with 50 mM sodium acetate pH 4.6. The pH ofthe eluate was increased to pH 7.4 by addition of 1 M phosphate bufferpH 6.6 (ratio eluate:buffer (v/v)=10:1) and 4 M NaOH. The proteinconcentration was 10 mg/ml, of which 78% is monomeric and maturerecombinant HSA.

[0054] Affinity Chromatography:

[0055] The HSA containing Q-sepharose eluate was contacted with2-mercaptodecanoic acid coupled to epoxy-activated sepharose 6FF by adiglycidylether (E-C10-C10-sepharose) as described in Example 1. Theaffinity absorbent was equilibrated with 100 mM sodium phosphate bufferpH 7.4. After binding the recombinant HSA to the absorbent (load: 22 gof protein per liter gel) the column was washed with the equilibrationbuffer. The HSA was eluted with 100 mM sodium phosphate pH 7.4containing 75 mM sodium caprylate. The HSA was desalted and concentratedby ultrafiltration using a filter with a cut-off of 30,000 D. Thepurified HSA had a protein concentration of 55 mg/ml, of which more than99.9% was monomeric and mature HSA.

[0056] In this example the recovery of HSA was 50%. No contaminants andonly trace amounts of degradation products could be detected by SDS-PAGE(FIG. 1), HPLC-SEC (FIGS. 2+3), HPLC-IEC (FIGS. 4+5) and IEF (FIG. 6).The purity according to HPLC-SEC was found to be 99.3%. Correction forthe salt related peak at 24.77 gives a purity of >99.91%. In fact, usingimmunoblotting techniques the purity of the purified HSA was estimatedto be at least 99.95%. For this technique polyclonal antibodies wereraised against proteins, which were purified from a blank K. lactisfermentation broth according to the method described in this example.The level of endotoxin was lower than 1 endotoxin unit per ml 5% (w/v)recombinant HSA concentrate. Endotoxin units are related to thereference standard (RSE) delivered by the FDA, the so-called EC-5.Endotoxin was mainly removed during the Q-sepharose column step, seeTable 1. TABLE 1 removal of endotoxin during purification of recombinanthuman serum albumin Composition Total endotoxin units K. lactissupernatant 115,000 effluent Q-sepharose 113,000 eluate Q-sepharose 59effluent affinity-matrix 41 eluate affinity-matrix 41 end product (5.5%(w/v) HSA) 17

Example 3A Purification of Recombinant HSA from a Clarified FermentationBroth by Q-Sepharose and E-C10-C8-Affinity Chromatography

[0057] Fermentation, filtration and ultrafiltration: performed asdescribed in Example 2.

[0058] Q-sepharose chromatography: performed as described inEP-A-0319067.

[0059] The collected supernatants were concentrated by ultrafiltrationusing a filter with a cut-off of 30,000 D. The pH of the retentate wasadjusted to pH 7.4. The concentration of monomeric HSA was 14 mg/ml.

[0060] Affinity Chromatography with E-C10-C8-Matrix:

[0061] The concentrated Q-sepharose eluate was added to C8(2-mercaptooctanoic acid) coupled to epoxy-activated sepharose 6FF by adiglycidylether. The affinity absorbent was equilibrated and washedafter absorption with 100 mM sodium phosphate buffer 7.4. The monomericHSA and degradation products could be eluated by 100 mM sodiumcaprylate. Intact HSA and degradation products could be separated byusing a gradient of 0-100 mM sodium caprylate in 100 mM sodium phosphatebuffer pH 7.4. Binding of HSA and degradation products to theE-C10-C8-matrix was less strong than binding to the E-C10-C10-matrix asdescribed in Example 2.

Example 3B Purification of Recombinant HSA from a Clarified FermentationBroth by Q-Sepharose and E-C3-C12-Affinity Chromatography

[0062] Fermentation, filtration, ultrafiltration and Q-sepharosechromatography: performed as described in Example 3A.

[0063] Affinity chromatography with E-C3-C12-matrix:

[0064] The purification of HSA was performed as described in Example 3A,except 2-mercaptododecaoctanoic acid was coupled to epoxy-activatedsepharose 6FF by C3-ether. Bound monomeric and mature HSA, degradationproducts of HSA and other contaminations could not be eluted by 100 mMsodium caprylate in 100 mM sodium phosphate buffer pH 7.4. However,elution was possible with 1% SDS. Separation of intact HSA and otherproteins was not possible.

1. A method for producing purified serum albumin, prepared byfermentation using transformed microorganisms or cell lines made byrecombinant DNA techniques comprising: starting with a clarifiedfermentation broth or cell lysate comprising said serum albumin; ifnecessary, changing the pH of the product medium to about physiologicpH; incubating the medium with an anion-exchange medium and eluting theserum albumin by washing with a buffer solution of acid pH; ifnecessary, changing the pH of the product medium to about physiologicpH; chromatographing said concentrated medium with a lipophilic immobilephase comprising a carrier coupled to a 2-mercapto or 2-hydroxy C4-C14alkanoic acid, or salt or ester thereof, and eluting the albumin byadding as desorbens a water soluble lipid anion to the aqueous phase;and isolating purified serum albumin substantially free of contaminantsfrom said microorganisms or cell lines.
 2. A method according to claim1, wherein said carrier has been activated by an epoxide compound.
 3. Amethod according to claim 2, wherein an agarose carrier has beenactivated by an epoxide compound of the following structure:

n and m are each, individually, 0-10, with the proviso that if x=halo,n=1.
 4. A method according to claim 3, wherein the epoxyether isdiglycidylether (viz. n=1, and

or epichlorohydrin (viz. X=chloro).
 5. A method according to any one ofthe preceding claims, including the additional step of dialysing saidpurified serum albumin.
 6. A method according to any one of thepreceding claims, wherein said serum albumin is human serum albumin. 7.A chromatography carrier coupled to a C4-C14 2-mercapto or 2-hydroxyalkanoic acid.
 8. An epoxy-activated carrier according to claim
 7. 9. Acomposition of isolated serum albumin, more than 99.9% pure,particularly more than 99.95% pure.
 10. A composition of isolated serumalbumin according to claim 9 for use as a medicament.